Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.21.1 (chymotrypsin)
10,938 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We synthesized five peptides homologous to the potentially antigenic positions alpha(214-226), alpha(430-443), alpha(415-443), beta(241-256), and beta(412-431) of the porcine brain tubulin sequences. These peptides were successfully employed to raise tubulin-cross-reactive antibodies. The antibodies are specific of the regions of tubulin spanned by the peptides. They react specifically with the tubulin bands in immunoblots and with microtubules in immunofluorescence assays of cytoskeletons. The peptides of the C-terminal regions have also been employed to localize determinants recognized by two available monoclonal antibodies to tubulin in the positions alpha(415-430) and beta(412-431), respectively. In a first application of the anti-peptide antibodies, we have mapped the fragments of limited proteolysis of purified calf brain tubulin by trypsin, chymotrypsin, papain, thermolysin, subtilisin, and protease V8 from Staphylococcus aureus. Thirty-seven peptides have been identified, of which 32 have been unequivocally aligned into the tubulin sequences on the basis of their antigenic reactivity. There are three major, well-defined zones of preferential cleavage by the proteases: the C-termini and two internal zones in each chain. C-Terminal cleavages of both chains by subtilisin do not remove the antigenic reactivity of the zones alpha(415-430) and beta(412-431). C-Terminal cleavages by protease V8 are preferential of beta-tubulin. All six proteases tested cleave alpha- and/or beta-tubulin at one or both of the internal zones. These zones are located roughly at one-third and two-thirds of the chain length in both subunits. Therefore, a model of the tubulin monomers is proposed which consists of three major, proteolytically defined, compact regions (N-terminal, middle, and C-terminal thirds) and the cleavable zones. This model is discussed with the tubulin structural information presently available.
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PMID:Tubulin structure probed with antibodies to synthetic peptides. Mapping of three major types of limited proteolysis fragments. 245 62

Two cystatins were purified from tissue extract of bovine brain by alkaline treatment, acetone fractionation, gel chromatography on Sephadex G-75, and affinity chromatography on S-carboxymethyl-papain-Sepharose. One of the inhibitors had a relatively high molecular mass, 25 kDa (HMM-cystatin) with pI 4.7, and the other, 11 kDa (LMM-cystatin) with pI 5.23. Both inhibitors showed considerable stability at pH 2 and 80 degrees C. The cystatins inhibited papain, ficin, and cathepsins B and H, but not trypsin, chymotrypsin, thermolysin, nagarse, and cathepsin D. Ki values for the complexes of papain and the inhibitors were estimated to be 2.8 x 10(-10) M for HMM-cystatin and 1.3 x 10(-9) M for LMM-cystatin. Both purified cystatins prevented degradation of substance P by soluble fraction and lysosomal extract obtained from synaptosomes, but did not suppress the cleavage of the peptide by synaptosomal plasma membranes.
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PMID:Cystatins from bovine brain: purification, some properties, and action on substance P degrading activity. 245 27

The primary structure of Pseudomonas cytochrome c peroxidase is presented. The intact protein was fragmented with cyanogen bromide into five fragments; partial cleavage was observed at a Met-His bond of the protein. The primary structure was established partly by automatic Edman degradations, partly by manual sequencing of peptides obtained with trypsin, thermolysin, chymotrypsin, pepsin, subtilisin and Staphylococcus aureus V8 endopeptidase. The order of the cyanogen bromide fragments was further confirmed by overlapping peptides obtained by specific cleavage of the whole protein. Pseudomonas cytochrome c peroxidase consists of 302 amino acid residues giving a calculated Mr of 33690.
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PMID:The primary structure of Pseudomonas cytochrome c peroxidase. 254 94

Gelsolin was cleaved by chymotrypsin or thermolysin into an N-terminal Mr 45,000 fragment (45N) and a C-terminal Mr 38,000 fragment (38C). The N-terminal half was further cleaved into two fragments with Mr 17,000 (17N) and Mr 28,000 (28N). These fragments were complexed with actin and cross-linked with 1-ethyl-3-[3-(dimethylamino)prophyl]carbodiimide (EDC) to introduce covalent bonds into their contact sites. The location of these bonds was mapped along the actin sequence by end-label fingerprinting with highly sensitive probes for the N- and C-termini of actin. The mapping studies revealed that two gelsolin N-terminal fragments (17N and 28N) were cross-linked with the actin C-terminal segment. The result indicates that the actin N- and C-terminal segments are in the binding site of gelsolin.
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PMID:End-label fingerprintings show that the N- and C-termini of actin are in the contact site with gelsolin. 254 8

The antigenic regions of the type II regulatory subunit of cAMP-dependent kinase from bovine heart have been correlated with the previously established domain structure of the molecule. Immunoblotting with both serum and monoclonal antibodies of fragments generated by limited proteolysis or chemical cleavage of the R-subunit established that the major antigenic sites were confined to the amino-terminal portion of the polypeptide chain (residues 1-145). Radioimmunoassays using two different antisera suggested that one or more of the high affinity serum antibody recognition sites were further restricted to residues 91-145. This amino-terminal portion of the R-subunit includes the hinge region which is particularly sensitive to proteolysis, allowing the R-subunit to be cleaved readily into a COOH-terminal domain which retains the cAMP-binding sites and an NH2-terminal fragment which appears to be the major site for interaction of the R-subunits in the native dimer. Monoclonal antibodies that recognized determinants on both sides of this hinge region were characterized and their specific recognition sites localized. Accessibility of antigenic sites in the holoenzyme in contrast to free R2 was compared. Although cAMP did tend to slightly increase the affinity of the holoenzyme for one of the monoclonal antibodies, all of the antigenic sites clearly were exposed and accessible in the holoenzyme. Furthermore, despite the presumed close proximity of antigenic sites to interaction sites between the R- and C-subunits, in no case did binding of antibody to the holoenzyme promote dissociation of the complex. The fact that the monoclonal antibodies would precipitate holoenzyme as well as free R2 was used to ascertain the importance of specific amino acid residues in the interaction of the R- and C-subunits. cAMP-binding domains were isolated following limited proteolysis with chymotrypsin and thermolysin. These fragments differed by only three amino acid residues at the NH2-terminal end. U of these fragments in conjunction with immunoadsorption established that the chymotryptic fragment, which contained the Asp-Arg-Arg preceding the site of autophosphorylation, was capable of forming a stable complex with the C-subunit. In contrast, the thermolytic fragment which differed by only those three residues no longer complexed with the C-subunit, indicating that the arginine residues not only contribute to the specificity of the phosphorylation site but also are an essential component for energetically stabilizing the holoenzyme complex.
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PMID:Monoclonal antibodies as probes for functional domains in cAMP-dependent protein kinase II. 257 46

The proteolytic cleavage of Chlamydia trachomatis LGV-434 surface proteins and resultant effects on infectivity and association with cultured human epithelial (HeLa) cells have been examined. Of several proteases examined, trypsin, chymotrypsin, and thermolysin extensively cleaved the chlamydial major outer membrane protein (MOMP). Two proteases, trypsin and thermolysin, cleaved the MOMP to the extent that monomeric MOMP was not detectable by immunoblotting with monospecific polyclonal antibodies. In the case of thermolysin, not even antigenic fragments were detected. Surprisingly, infectivity toward HeLa cells was not diminished. In addition, the association of intrinsically 14C-radiolabeled elementary bodies (EBs) with HeLa cells or their dissociation by proteinase K was not measurably affected by prior trypsinization of the EBs. Trypsinization of lactoperoxidase surface-iodinated elementary bodies demonstrated that most of the 125I-labeled surface proteins were cleaved. In all cases, however, a number of proteolytic cleavage fragments remained associated with the EB surface after surface proteolysis. When trypsinized EBs were electrophoresed under nonreducing conditions and immunoblotted with either polyclonal or type-specific monoclonal MOMP antibodies, MOMP was found in a large oligomeric form that failed to enter the polyacrylamide stacking gel. Additionally, trypsinized viable EBs bound radioiodinated type-specific MOMP monoclonal antibody as efficiently as did the control nontrypsinized organisms. Taken together, the findings indicate that although the MOMP is highly susceptible to surface proteolysis, the supramolecular structure of the protein on the EB surface is apparently maintained by disulfide interactions. Thus, if surface-exposed chlamydial proteins are involved in the initial interaction of chlamydiae with eucaryotic cells, the functional domains of these proteins which mediate this interaction must be resistant to proteolysis and remain associated with the EB surface.
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PMID:Effect of proteolytic cleavage of surface-exposed proteins on infectivity of Chlamydia trachomatis. 258 Jul 94

A 39 kD protein has been extracted from barley flour with 0.1 M monothioglycerol at pH 5.0 and purified by (NH4)2SO4-precipitation, anion exchange and molecular sieve chromatography. It is an N-terminally blocked, non-glycosylated, single-chain protein present in at least two molecular forms of isoelectric points 5.18 and 5.22. The amino acid composition and partial sequence analysis reveal a relationship to barley endosperm Z protein which belongs to the serpin superfamily. The 39 kD protein inhibits alpha-chymotrypsin while little or no effect could be demonstrated on trypsin, subtilisin, proteinase K, S. aureus V8 protease, thermolysin or two malt thiol endoproteinases. The 39 kD protein is immunochemically related to the major protein component in beer.
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PMID:A 39 kD barley seed protein of the serpin superfamily inhibits alpha-chymotrypsin. 263 81

A glutaredoxin was purified from rabbit bone marrow, and its amino acid sequence was determined by high performance tandem mass spectrometry. The sequences of peptides generated by digestion with trypsin alone or in combination with thermolysin were determined from their collision-induced dissociation (CID) mass spectra. Alignment of these sequences and additional sequence information were obtained from the collision-induced dissociation mass spectra of peptides obtained from digestion of the intact protein with Staphylococcus aureus V8 protease and alpha-chymotrypsin. The resulting sequence of 106 amino acids is as follows: Ac-Ala-Gln-Glu-Phe-Val-Asn-Ser-Lys-Ile-Gln-Pro-Gly-Lys-Val-Val-Val-Phe- Ile-Lys-Pro-Thr-Cys-Pro-Tyr-Cys-Arg-Lys-Thr-Gln-Glu-Ile-Leu-Ser-Glu-Leu- Pro-Phe - Lys-Gln-Gly-Leu-Leu-Glu-Phe- Val-Asp-Ile-Thr-Ala-Thr-Ser-Asp-Met-Ser-Glu-Ile- Gln-Asp-Tyr-Leu-Gln-Gln-Leu-Thr-Gly-Ala-Arg- Thr-Val-Pro-Arg-Val-Phe-Leu-Gly-Lys-Asp-Cys-Ile- Gly-Gly-Cys-Ser-Asp-Leu-Ile-Ala-Met-Gln-Glu-Lys- Gly-Glu-Leu-Leu-Ala-Arg-Leu-Lys-Glu-Met-Gly- Ala-Leu-Arg-Gln. This glutaredoxin strongly resembles the corresponding calf and pig proteins (known as glutaredoxin and thioltransferase, respectively) with respect to its primary structure and enzymatic activity as a GSH:disulfide thioltransferase, an activity also found for the glutaredoxin from Escherichia coli. However, rabbit glutaredoxin was not active as a hydrogen donor for the reduction of ribonucleotides in the presence of the ribonucleotide reductases from rabbit bone marrow, Lactobacillus leichmannii, and Corynebacterium nephridii.
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PMID:Glutaredoxin from rabbit bone marrow. Purification, characterization, and amino acid sequence determined by tandem mass spectrometry. 268 77

Bovine articular type II collagen was prepared by limited pepsin digestion, differential salt fractionation and carboxymethylcellulose chromatography. Cyanogen bromide digestion of purified type II collagen alpha chains yielded twelve distinct peptides designated CB1-12. The peptide alpha 1(II)-CB11 was isolated by carboxymethylcellulose chromatography and Sephadex G-75S gel filtration. Automated Edman degradation together with chymotrypsin, thermolysin and trypsin digestion enabled identification of its complete amino acid sequence. Compared with type I and type III collagen, the data show similarity with alpha 1(I)-CB8 and alpha 1(III)-CB6-1-8-10-2 peptides, respectively. The peptide is located within residues 124-402 of the alpha 1(II) collagen chain and with its identification, now extends the known amino acid sequence of bovine type II cartilage collagen to 660 amino acid residues including alpha 1(II)-CB1-2-6-12-11-8-10 (partial). This corresponds to alpha 1(I)-CB0-1-2-4-5-8-3-7 (partial; 1-660) and alpha 1(III)-CB3A-3B-3C-7-6-1-8-10-2-4-5 (partial; 1-660) of bovine alpha 1(I) and alpha 1(III) collagen chains.
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PMID:Covalent structure of collagen. Amino acid sequence of an arthritogenic cyanogen bromide peptide from type II collagen of bovine cartilage. 271 76

Radiolabeled insulin was affinity cross-linked to purified insulin receptor with six separate bifunctional N-hydroxysuccinimide esters of different lengths. Results were qualitatively identical for each cross-linker in that insulin was predominantly cross-linked through its B chain to the receptor's alpha subunit. The maximum efficiencies of cross-linking were 10-15% for the most effective reagents, and this value was dependent upon the concentration and length of the cross-linker. In an effort to locate the cross-linking site, monoiodoinsulin was cross-linked to affinity-purified insulin receptor with disuccinimidyl suberate. Limited proteolysis of the hormone/receptor adduct with Staphylococcus aureus V8 protease, chymotrypsin, or thermolysin in an SDS-containing buffer rapidly generated a 55-kDa, insulin-labeled fragment as shown by SDS-polyacrylamide gel electrophoresis. We reported earlier that the 55-kDa chymotryptic fragment contained multiple internal disulfide bonds as evidenced by its shifting mobility on an SDS gel after dithiothreitol treatment [Boni-Schnetzler et al. (1987) J. Biol. Chem. 262, 8395-8401]. Here we show that the 55-kDa fragment is also formed by proteolysis of the receptor in the absence of prior insulin cross-linking. This fragment was prepared in amounts sufficient for sequence analysis and was purified by passage successively over gel permeation and reverse-phase HPLC columns. The sequence of the fragment's amino terminus corresponds to that of the amino terminus of the receptor's alpha subunit. This fragment also reacts with an antibody raised against a synthetic peptide corresponding to residues 242-253 of the receptor's alpha subunit.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Isolation of a proteolytically derived domain of the insulin receptor containing the major site of cross-linking/binding. 274 47


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